Harold Gainer

GABA in the mammalian suprachiasmatic nucleus and its role in diurnal rhythmicity

Mammals manifest circadian behaviour timed by an endogenous clock in the hypothalamic suprachiasmatic nucleus (SCN). Considerable progress has been made in identifying the molecular basis of the circadian clock, but the mechanisms by which it is translated into cyclic firing activity, high during the day and low at night, are still poorly understood. GABA (γ-aminobutyric acid), a common inhibitory neurotransmitter in the central nervous system, is particularly densely distributed within the SCN, where it is located in the majority of neuronal somata and synaptic terminals. Using an in vitro brain-slice technique, we have now studied the effect of bath-applied GABA on adult SCN neurons at various times of the day. We find that GABA acts as an inhibitory neurotransmitter at night, decreasing the firing frequency; but during the day GABA acts as an excitatory neurotransmitter, increasing the firing frequency. We show that this dual effect, which is mediated by GABAA receptors, may be attributed to an oscillation in intracellular chloride concentration. A likely explanation is that the amplitude of the oscillation in firing rate, displayed by individual neurons, is amplified by the dual effect of GABA in the SCNs GABAergic network.

Peptide Regulation of Bursting Pacemaker Activity in a Molluscan Neurosecretory Cell

Vasopressin and related peptides (10-9 to 10-6 molar) induced bursting pacemaker potential activity and altered the current-voltage relations of the membrane in a specific molluscan neurosecretory cell. These effects long outlasted the period of application of the peptides. Sensitivity of the cell to these peptides was primarily localized on the axon hillock region. The observed effects do not resemble conductance changes evoked by conventional neurotransmitters, but rather suggest a membrane regulatory role for these peptides, and thus may be indicative of a new form of information transfer in the nervous system.

Studies on bursting pacemaker potential activity in molluscan neurons. III. Effects of hormones

Vertebrate peptides and hormones have been appled to a number of identified neurosecretory and ono-neurosecretory cells in two molluscan preparations. Active peptide hormones included vasopressin and analogues. Active steriod hormones included aldosterone and hydrocortisone. Peptide effects were present at 10-9 M concentration of peptide, were confined to two neurosecrotory cells and consisted of long lasting changes in the membrane properties of these cells (characterized either by the initiation or potentiation of bursting pacemaker potential activity in these cells). The regulatory changes in membrane properties induced by the peptides were unlike the transient conductance changes produced by conventional neurotransmitters. Steroid effects were observed at 10-6M concentration of steroid and consisted of an increase in membrane potential and conductance which was dependent on the species of divalent cations present. The net effect of peptide activation would be to increase the release of neurosecretory material form the cell terminals, while the net effect of the steroids would be to decrease the release of this material. The results obtained with these invertebrate preparations may serve to describe new forms of cellular communication in the nervous system whereby peptides and steroids modulate electrical activity.

Electrophysiological behavior of an endogenously active neurosecretory cell

Summary An identified neurosecretory cell in the snail, cell 11 in Otala lactea , has been shown to have patterned impulse activity of endogenous origin. Several lines of evidence supporting this view are: hyperpolarization of the cell revealed no EPSPs driving the cell, the cell continued to act as a bursting pacemaker in concentrations of magnesium ion sufficient to block all synaptic activity, and the isolated soma of the cell exhibited spontaneous bursting behavior. While the action potential was due to increases in sodium and calcium conductances, the pacemaker potential was dependent only on the sodium ions in the medium. Stimulation of peripheral nerves evoked only IPSPs in cell 11 and, therefore, it was suggested that the cells secretory activity was modulated primarily by inhibitory processes. The axon of cell 11 runs in the visceral nerve and terminates specifically in the auricle of the snails heart. The activity of cell 11 did not appear to effect the rate or strength of the heartbeat, which suggested that this neurohemal location of termination was specifically related to the cells secretory activity.

Vasopressin Coexists in Half of the Corticotropin-Releasing Factor Axons Present in the External Zone of the Median Eminence in Normal Rats

Electron-microscopic immunocytochemistry of serial thin sections showed that, in normal rats, approximately 50% of corticotropin-releasing factor (CRF) axons in the external zone of the median eminence contained vasopressin (AVP) and AVP precursor-derived peptides coexistent in the same secretory vesicles as CRF. The other 50% of the CRF axons contained little or no detectable AVP or AVP precursor-derived peptides. Differential activation of these subpopulations of CRF axons could provide a mechanism by which levels of CRF and AVP in portal plasma could be varied independently.

Neurophysin biosynthesis: conversion of a putative precursor during axonal transport

[35S]Cysteine injected adjacent to the supraoptic nucleus of the rat is rapidly incorporated into a 20,000-dalton protein that, in time, is converted to a 12,000-dalton labeled protein, neurophysin. This putative precursor of neurophysin appears to be synthesized in the supraoptic nucleus and transformed to neurophysin and related peptides during axonal transport to the neurohypophysis.

PSD-95 Is Required to Sustain the Molecular Organization of the Postsynaptic Density

PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.

Pentobarbital: selective depression of excitatory postsynaptic potentials.

The effects of pentobarbital (Nembutal) on synaptic transmission and postsynaptic potentials were studied by the use of several invertebrate preparations. Pentobarbital selectively and reversibly depressed both excitatory postsynaptic potentials and sodium-dependent postsynaptic responses to putative excitatory transmitters without affecting either inhibitory postsynaptic potentials or chloride- and potassium-dependent postsynaptic responses to putative transmitters. A selective depression of postsynaptic excitatory events was also observed with other central nervous system depressants (ethanol, chloroform, chloralose, diphenylhydantoin, and urethane). The results suggest that central and peripheral depression observed during general anesthesia is due to a selective depression of excitatory synaptic events.

Structure of mouse vasopressin and oxytocin genes.

Mouse vasopressin (VP) and oxytocin (OT) genes were isolated from a genomic library and the nucleotide sequences of the two genes were determined. The two genes have similar three exon structures and a high similarity in the part of exon 1 encoding vasopressin or oxytocin nonapeptide and in exon 2 encoding the central core of neurophysin. They are linked together in a tail to tail orientation separated by a short 3.5 kb intergenic sequence and are transcribed from opposite strands. Both genes have a single transcription initiation site downstream from a TATA-like sequence and a single polyadenylated transcript of about 760 bp for the vasopressin mRNA and about 700 bp for the oxytocin mRNA.

Single Cell Reverse Transcription-Polymerase Chain Reaction Analysis of Rat Supraoptic Magnocellular Neurons: Neuropeptide Phenotypes and High Voltage-Gated Calcium Channel Subtypes

Magnocellular neurosecretory cells (MNCs) in the hypothalamo-neurohypophysial system that express and secrete the nonapeptides oxytocin (OT) and vasopressin (VP) were evaluated for the expression of multiple genes in single magnocellular neurons from the rat supraoptic nucleus using a single cell RT-PCR protocol. We found that all cells representing the two major phenotypes, the OT and VP MNCs, express a small, but significant, amount of the other nonapeptide’s messenger RNA (mRNA). In situ hybridization histochemical analyses confirmed this observation. A third phenotype, containing equivalent amounts of OT and VP mRNA, was detected in about 19% of the MNCs from lactating female supraoptic nuclei. Analyses of these phenotypes for other coexisting peptide mRNAs (e.g. CRH, cholecystokinin, galanin, dynorphin, and the calcium-binding protein, calbindin) generally confirmed expectations from the literature, but revealed cell to cell variation in their coexpression. Our results also show that the high voltage...